Probes Synthesis for Animal Use

Synthesis of precursor PSMA-1-DOTA: PSMA-1-DOTA was synthesized on solid phase by Fmoc-chemistry as reported previously at 0.1 mmol scale [26]. After Glu-CO-Glu’-Amc-Ahx-dGlu-dGlu-dGlu-Lys(Mtt)-rink amide resin was built on the resin, the Mtt group on Glu-CO-Glu’-Amc-Ahx-dGlu-dGlu-dGlu-Lys(Mtt)-rink amide resin was removed by 2% TFA in dichloromethane. The resin was washed with 2% DIPEA in DMF and resuspend in DMF, then 3 equivalents of DOTA NHS ester (Macrocyclics) and 3 equivalents of DIPEA was added. After the reaction was completed as indicated by Kaiser test (AAPPTEC), PSMA-1-DOTA was cleaved from the resin by trifluoroacetic acid (TFA)/water/triisopropylsilane (950:25:25) and purified by preparative high performance liquid chromatography (HPLC) using Luna 5 µ C18(2) 100 column (250 mm × 10 mm × 5 μm, Phenomenex, Torrance, CA) at a flow rate of 2.5 ml/min. The gradient used was 10%—40% acetonitrile against 0.1% trifluoroacetic acid over 30 min. Purity: > 98%. Retention time: 18.9 min (Figure S1A). EI-MS and MALDI-TOF–MS: C62H100N14O27, calculated: 1474.69; found: 1474.38 (Figure S1B-C). 1H NMR (500 MHz, D2O): δ (ppm)m 0.96 (m, 2H), 1.33 (m, 2H), 1.41 (m, 4H), 1.48 (m, 4H), 1.50 (m, 1H), 1.60 (m, 2H), 1.82 (m, 6H), 1.97 (m, 6H), 2.16 (m, 7H), 2.32 (t, 2H), 2.39 (t, 2H), 2.50 (m, 8H), 3.05 (m, 2H), 3.16 (t, 4H), 3.33 (br, 16H), 3.80 (br, 8H), 4.18 (m, 1H), 4.26 (m, 4H), 4.38 (m, 1H) (Figure S1D-F).

Synthesis of cold Ga-PSMA-1-DOTA: PSMA-1-DOTA (1.4 mg, 0.97 μmol) was dissolved in 400 μL of 0.2 M sodium acetate (pH 3–4), to which fivefold excess gallium nitrate (1.25 mg, 4.9 μmol) in 400 μL of 0.2 M sodium acetate (pH 3–4) was added. The reaction mixture was stirred at 90 °C for 1 h and went through HPLC for purification. The HPLC condition was the same as the purification of PSMA-1-DOTA. Yield: 1.1 mg, 78%. Purity: > 98%. Retention time: 18.7 min. EI-MS and MALDI-TOF–MS: C62H98GaN14O27, calculated: 1540.60; found: 1541.61 (Figure S2).

Gallium-68 radiolabeling: The synthesis of [68 Ga]Ga-PSMA-1-DOTA was carried out based on a protocol described by Scintomics GmbH for routine production of [68 Ga]Ga-PSMA-11. This protocol was improved and validated at University Hospital (UH) PET radiopharmacy, and generator elution mode was set up in accordance with Eckert & Ziegler directives for IGG-100 generator elution. The entire synthesis, including elution of radioactive gallium from the generator with 0.1 M hydrochloric acid, conjugation with PSMA-1 peptide, purification and formulation of the final product are automated by Scintomics GRP® synthesizer and controlled by the software using GMP-manufactured single-use sterile cassettes. Gallium-68 was produced at University Hospital (UH) PET Radiochemistry facility using a 50 mCi IGG100-50 M 68Ge/68 Ga generator purchased from Eckert & Ziegler Isotope Products. After the program started, the C18 cartridge was preconditioned with ethanol (5 mL) and washed with water (20 mL). The 68 Ga was eluted from 68Ge/68 Ga generator with 5 mL of hydrochloric acid (0.1 M) and the eluent was diluted with 6 mL water in the syringe. This solution was passed through the strong cationic exchange cartridge in proton cycle (PS-H+) where 68 Ga was retained, and subsequently eluted in the next step with sodium chloride solution (5 M, 1.7 mL) into the pre-heated reactor containing 20 µg PSMA-1 precursor peptide (added as 20 µL of stock solution with concentration of 1 mg/mL) and HEPES buffer (1.5 M, pH 4.5, 1.5 mL). The labeling took 5 min at 90 °C (temperature set for the heater). After labelling step, the reactor content was transferred onto the C18 Plus Light SPE-cartridge, where the labeled [68 Ga]Ga-PSMA-1-DOTA was retained while the ionic 68 Ga, HEPES, unreacted precursor and salts are washed out with water for injection (8 mL). The labeled [68 Ga]Ga-PSMA-1-DOTA product was eluted from the C18 cartridge with mixture of water for injection/ethanol (1/1, v/v, ~ 2 mL) through the 0.22 µm membrane sterile filter into the final product vial. Finally, the product is diluted with ~ 13 mL PBS through the same sterile 0.22 µm membrane filter into the final product vial. Samples were then aseptically removed for quality control testing. Radio-TLC and Radio-HPLC were performed using the same analysis for [68 Ga]Ga-PSMA-11 to confirm the success of radiolabeling. Similarly, [68 Ga]Ga-PSMA I&T was also synthesized using the Scintomics system for comparison scans. The products were diluted with ~ 13 mL sterile PBS in the final product vial. Samples were then aseptically removed for quality control testing.

Lutetium-177 radiolabeling: A stock solution of 177Lu (Eckert & Ziegler) in 0.04 M HCl was prepared in a clean reaction vial. The 177Lu was then diluted with up to 250 µL 0.25 M sodium acetate, pH 5.5. A solution of 25–250 ug PSMA-1-DOTA (1 mg/mL in 0.25 M sodium acetate, pH 5.5) was then added to the 177Lu solution. The reaction was allowed to mix for 60 min at 90 ˚C. The reaction was checked via radio-TLC and if the crude yield was below 90%, then an additional 25 µg PSMA-1-DOTA (1 mg/mL in 0.25 M sodium acetate, pH 5.5) was added with mixing continued for an additional 30 min. The crude reaction was then sterile filtered using a 0.22 µm sterile filter.

The product was analyzed by Radio-TLC on Macherey–Nagel silica gel 60 plates eluted with 100 mM EDTA in 100 mM PBS, pH 7.2 (Rf: [177Lu]Lu-PSMA-1-DOTA 0.0, 177Lu 0.6) and by RP-HPLC with a method developed using an Agilent 1090 HPLC equipped with a radiochemical detector. Specifically, the sample was analyzed on a Phenomenex Luna Omega C18 5 µm column with linear gradient of 100% of solvent A to 100% solvent B in 17 min. Solvent A is 0.1% TFA in water and solvent B is 0.1% TFA in acetonitrile. [177Lu]Lu-DOTA-PSMA-1 has a retention time of 11.5 min. Free 177Lu has a retention time of 4.5 min, Fig. 7S.

Cell Lines

Retrovirally transformed PSMA positive PC3pip cells were obtained from Dr. Michel Sadelain in 2000 (Laboratory of Gene Transfer and Gene Expression, Gene Transfer and Somatic Cell Engineering Facility, Memorial-Sloan Kettering Cancer Center, New York, NY). The cells were last checked and authenticated by flow sorting and western blot in 2023. Cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2 under a humidified atmosphere.

Competition Binding Assay

Competition binding assay was performed as described previously using N-[N-[(S)−1,3-dicarboxypropyl]carbamoyl]-S-[3H]-methyl-L-cysteine (3H-ZJ24) (GE Healthcare Life Sciences, Pittsburg, PA) [26, 27]. (S)−2-(3-((S)−5-amino-1-carboxypentyl)ureido)pentanedioic acid (ZJ24) was custom made by Bachem Bioscience Inc. (Torrance, CA). PSMA-11 and PSMA-I&T was purchased from MedChemExpress. PC3pip cells (5 × 105) were incubated with different concentrations of PSMA ligands in the presence of 12 nM 3H-ZJ24 in 50 mM Tris buffer (pH 7.5) for 1 h. The cells were then washed 3 times with cold phosphate-buffered saline (PBS) and the radioactivity retained by cells was measured by scintillation counting. The concentration required to inhibit 50% of binding (IC50) was determined by GraphPad Prism 3.0

Mouse Subcutaneous Xenograft Model

All animal procedures were performed according to Case Western Reserve University’s Institutional Animal Care and Use Committee (IACUA) approved protocols. Male athymic nude (Foxn1/nu/Foxn1/nu) mice 6–8 weeks old (Jackson Laboratory) were used to generate tumor xenografts. Upon arrival, mice were maintained in a 12-h light/12 -hour dark cycle with free access of water and food. PC3pip cells (1 × 106) were suspended in 100 μL of Matrigel/PBS mixture (1:1) and injected to the right flank of the mice. Mice were then observed every other day and were ready to use when the tumor size reached about 100 mm3.

Fluorescence Imaging with PSMA-1-IR800

PSMA-1-IR800 was synthesized as previously reported [26]. Male athymic nude mice bearing PC3pip tumor were injected with 2 nmol of PSMA-1-IR800 via tale vein injection. Mice were imaged at 24-h post injection using a Curadel RP1 camera using the 800 nm channel. After in vivo imaging, mice were sacrificed and organs such as salivary gland, heart, liver, lung, spleen, kidneys and tumor were excised for ex vivo imaging.

MicroPET/CT imaging with 68 Ga-PSMA Agents

Male athymic nude mice (Foxn1/nu/Foxn1/nu, Jackson Laboratory) with or without subcutaneous PC3pip tumors were administered 7.4 MBq (200 μCi) of [68 Ga]Ga-PSMA-1-DOTA through tail vein injection. Five minute-static microPET scans were performed at 30 min and 1 h post injection using a Siemens Inveon microPET-CT scanner (Siemens Medical Solutions, Knoxville, TN). PET images were reconstructed using the default iterative reconstruction supplied by the vendor of Inveon. During imaging mice were under anesthesia with 2.0% isoflurane delivered in oxygen gas with a nose cone. Immediately after the micro-PET scan was complete, a CT scan was performed for better anatomic localization. Within a week, two other imaging sessions were carried out in 24- or 48-h intervals with the same mice injected with 7.4 MBq (200 μCi) of either [68 Ga]Ga-PSMA-11 or [68 Ga]Ga-PSMA I&T in 200 μL saline solution and similar microPET/CT scans were performed at 1 h post injection.

Regions of interests (ROIs) were drawn over PET/CT overlapped images to determine uptake in the parotid glands (left and right), lacrimal glands (left and right), kidneys (left and right) and PSMA-positive PC3pip xenograft tumors as well as muscle tissues (background). Standardized uptake values (SUVs, normalized by body weight) were calculated for quantification [28, 29]. Based on SUVmax values, tumor-to-organ ratios were calculated for the kidneys, salivary glands and muscle background for both [68 Ga]Ga-PSMA-11 and [68 Ga]Ga-PSMA-1-DOTA and are reported in Table 1.

Table 1 Tumor-to-Organ ratios comparing [68 Ga]Ga-PDMA-11 (PSMA-11) and [68 Ga]Ga-PSMA-1-DOTA (PSMA-1-DOTA)Tumor Inhibitory Activity of [177Lu]Lu-PSMA-1-DOTA

For treatment experiments, [177Lu]Lu-PSMA-1-DOTA was labeled by 3D Imaging, LCC with clinical grade [177Lu]Lu-PSMA-617 (Pluvicto, Novartis) used as positive control for RLT. Male athymic nude mice (Foxn1/nu/Foxn1/nu, Jackson Laboratory) with subcutaneous PC3pip tumors approximately 100 mm3 in size were randomly assigned to three groups of three mice each. On day 0, the mice were injected intravenously with either vehicle control (negative control) or received a single dose of 111 MBq (3 mCi) of [177Lu]Lu-PSMA-617 as a positive control. The third group of animals received a total of 111 MBq (3 mCi) of [177Lu]Lu-PSMA-1-DOTA administered in 4 doses (27.75 MBq each over 2 days). The second radioligand treatment was administered on day 13 with mice receiving a single dose of 111 MBq (3 mCi) for [177Lu]Lu-PSMA-1-DOTA and [177Lu]Lu-PSMA-617. Tumor volumes were measured every 3 to 4 days for 24 days following treatment.

Toxicity Studies of PSMA-1-DOTA

After synthesis and purification of PSMA-1-DOTA for compassionate human use, good laboratory practice (GLP) toxicity studies were performed by ATRC Aurigon Toxicological Research Center Ltd. (Hungary). Eight-week-old male NMRI mice (Charles River Laboratories) were used for the study. The study was a single dose extended study designed to assess immediate effects of PSMA-1-DOTA injection (main animals) and recovery from immediate effects after 15 days (recovery animals). The main animal group had 20 mice, in which, 10 mice received 0.9% normal saline as controls, and the other 10 mice received 1.4 mg/kg of PSMA-1-DOTA via intravenous (i.v.) injection. In the recovery animal group, five mice received 0.9% normal saline, and the remaining five received 1.4 mg/kg of PSMA-1-DOTA intravenously. The dose level was based on micro-dosing used on humans and was 1000X the maximum human micro-dose of 100 ug per dose assuming an average human body of 70 kg. After injection (Day 1) blood sampling was performed on Day 2 for main animals (20 animals) and on Day 15 for recovery animals (10 animals). Recovery animals were observed for a 15-day period. Necropsy was performed on day 2 for main animals and day 15 for recovery animals. Clinical chemistry and histopathology studies were performed.

“First in Human” Compassionate Use

[68 Ga]Ga-PSMA-1-DOTA was administered to a patient on a compassionate use basis. The production of [68 Ga]Ga-PSMA-1-DOTA was in compliance with the German Arzneimittegesetz (AMG), §13 2b, and the responsible regulatory authority (Government of Upper Bavaria) was duly notified. That is, [68 Ga]Ga-PSMA-1-DOTA was synthesized as described above at 0.4 mmol scale and the purity was confirmed by HPLC to be more than 98%. Written informed consent was obtained from the patient. 282 MBq (7.62 mCi) of [68 Ga]Ga-PSMA-1-DOTA was injected i.v. and a whole-body PET scan using a Siemens Biograph Vision with 2-min/bed from the top of the head to the thigh was taken at 1 h post-injection. PET images were reconstructed using the iterative OSEM with Time-of-Flight and resolution recovery ("TrueX") on the Biograph Vision system.